Cyclopamine (11-deoxojervine) is a steroidal alkaloid that occurs naturally in plants of the genera Veratrum, Zigadenus, Schoenocaulon1 and others. Its formula is C27H41NO2 with a molecular weight of 411.63. The structure of cyclopamine is shown in FIG. 1. 1 R. F. Keeler, Lipids 13 (1978) pp. 708-715.

An epidemic of sheep congenital deformities during the 1950's was eventually traced to the presence of the alkaloids jervine and cyclopamine in the wild lily Veratrum californicum upon which the sheep grazed2,3. It is now known that these teratogenic effects of jervine and cyclopamine are due to their specific inhibition of cellular responses to the Hedgehog (Hh) family of secreted growth factors4,5. In accordance with this general mechanism of hedgehog pathway activity, cyclopamine and related compounds have shown promise in the treatment of diverse cancers. These include for example brain tumors6, breast cancer7, ovarian cancer8, prostate cancer9, colorectal cancer10 and pancreatic cancer11. Although the primary focus has been on cancer, cyclopamine and its analogues are also being explored in a large number of other clinical conditions where a therapeutic effect can be obtained for a condition or disorder by inhibiting one or more aspects of Hedgehog pathway activity, including for example psoriasis12. 2 R. F. Keeler, Phytochemistry 7 (1968) pp. 303-306.3 R. F. Keeler and W. Binns, Phytochemistry 10 (1971) pp. 1765-1769.4 M. K. Cooper, J. A. Porter, K. E. Young and P. A. Beachy, Science 280 (1998) pp. 1603-1607.5 J. P. Incardona, W. Gaffield, R. P. Kapur and H. Roelink, Development 125 (1998) pp. 3553-3562.6 P. Sanchez, A. Ruiz i Altaba, Mech. Dev. 22 (2005) pp. 223-230.7 M. Katano, Cancer Lett. 227 (2005) pp. 99-104.8 X. Chen, A. Horiuchi, N. Kikuchi, R. Osada, J. Yoshida, T. Shiozawa, K. Konishi, Cancer Sci. 98 (2007) pp. 68-76.9 M. Mimeault, E. Moore, N. Moniaux, J. P. Henichart, P. Depreux, M. F. Lin, S. K. Batra, Int. J. Cancer 118 (2006) pp. 1022-1031.10 D. Qualtrough, A. Buda, W. Gaffield, A. C. Williams, C. Paraskeva, Int. J. Cancer 110 (2004) pp. 831-837.11 S. P. Thayer, M. Pasca di Magliano, P. W. Heiser, C. M. Nielsen, D. J. Roberts, G. Y. Lauwers, Y. P. Qi, S. Gysin, C. Fernandez-del Castillo, V. Yajnik, B. Antoniu, M. McMahon, A. L. Warshaw, M. Hebrok, Nature 425 (2003) pp. 851-856.12 S. Tas and O. Avci, Dermatology 209 (2004) pp. 126-131.
Cyclopamine was first isolated from V. grandiflorum in 196413 and later from V. californicum2,3. Cyclopamine was isolated in small amounts by extraction of alkanized ground plant roots with benzene, and subsequent purification by recrystallization in acetone/water and then methanol/water followed by silica gel column chromatography using a benzene/methanol mobile phase. This method results in very low recovery of the available alkaloids and cannot be easily scaled up. Recently, Oatis et al. 14 published a method for isolating cyclopamine from V. californicum by extracting kilogram-sized batches of biomass in a Soxhlet extractor using refluxing benzene for 14 hours. The benzene solution was then passed through a silica gel column and the trapped cyclopamine eluted using stepped gradient dicholormethane-isopropanol solutions. Cyclopamine-rich fractions were then re-chromatographed with ethyl acetate as the mobile phase and the resulting fractions triturated with acetone and recrystallized from ethanol-water to give purified cyclopamine. The final recovery was reported at approximately 55% of the available cyclopamine. This process is not scalable to large volumes. Benzene is a known carcinogen and dicholormethane, and other chlorinated hydrocarbons, are recognized as toxic and potentially carcinogenic and so it is desirable to avoid utilizing either in large scale processes. No other published methods for purifying cyclopamine from natural sources exist. 13 T. Masamune, T. Mori, M. Takasugi and A. Mural, Tetrahedron Lett. 16 (1964) pp. 913-917.14 J. E. Oatis, P. Brunsfeld, J. W. Rushing, P. D. Moeller, D. W. Bearden, T. N Gallien and G. Cooper, Chemistry Central Journal 2 (2008) p. 12.
The molecular complexity of cyclopamine suggests that a total synthesis from readily available raw material is not likely to be economically feasible. Preparation of pharmaceuticals and pharmaceutical compositions based on cyclopamine and its analogues and derivatives will therefore depend on a supply of the purified compound from natural sources. It is therefore of great interest to develop efficient methods of extracting, purifying and isolating cyclopamine from organic matter. No prior art exists for efficient, industrial-scale extraction, purification and isolation of cyclopamine. This invention provides such a method.